Project Summary The replication and transcription machineries function simultaneously and on the same DNA template. The coupling of these two processes leads to collisions that stall the replisome and promote mutagenesis. Our recent discovery suggests that collisions are particularly prevalent and disruptive during infections, and that their resolution is essential for the survival of bacterial pathogens inside eukaryotic hosts. Therefore, determining the molecular mechanisms underlying the detrimental consequences of collisions and their subsequent resolution is essential to our understanding of the fundamental principles governing bacterial pathogenesis. We recently determined that collisions promote R-loop formation and that these structures are primarily responsible for the detrimental outcomes of these encounters. Accordingly, we found that the highly conserved R-loop processing enzyme RNase HIII is essential for cells to tolerate severe collisions. Preliminary evidence I have gathered suggests that key virulence genes are collision hotspots and maybe particularly prone to R-loop formation. If so, then RNase HIII is a previously unappreciated yet critical virulence factor. Here, I propose to investigate this phenomenon using the model pathogen Listeria monocytogenes. Specifically, I will determine how collisions and subsequent RNase HIII activity impact pathogen replication inside eukaryotic cells. Using genome-wide deep sequencing approaches, I will map chromosomal hotspots of R-loop formation and the impact of these structures on DNA replication, as well as transcription, during pathogenesis. Furthermore, using deep mutational scanning, I will interrogate RNase HIII function in vivo. Altogether, this work will deepen our understanding of the basic biological mechanisms driving bacterial pathogenesis.